Topoisomerase III

ABSTRACT

Topoisomerase III polypeptides and DNA and RNA encoding such Topoisomerase III polypeptides and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such Topoisomerase III for the treatment of infection, particularly bacterial infections. Antagonists against such Topoisomerase III and their use as a therapeutic to treat infections, particularly bacterial infections are also disclosed. Also disclosed are diagnostic assays for detecting diseases related to the presence of Topoisomerase III nucleic acid sequences and the polypeptides in a host. Also disclosed are diagnostic assays for detecting polynucleotides encoding Streptococcal Topoisomerase El and for detecting the polypeptide in a host.

RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.08/949,584, filed Oct. 14, 1997 and U.S. Pat. No. 5,962,303, claimsbenefit of U.S. Provisional Application Ser. No. 60/028,603, filed Oct.15, 1996.

FIELD OF THE INVENTION

This invention relates, in part, to newly identified polynucleotides andpolypeptides; variants and derivatives of these polynucleotides andpolypeptides; processes for making these polynucleotides and thesepolypeptides, and their variants and derivatives; agonists andantagonists of the polypeptides; and uses of these polynucleotides,polypeptides, variants, derivatives, agonists and antagonists. Inparticular, in these and in other regards, the invention relates topolynucleotides and polypeptides of bacterial “Topoisomerase III”.

BACKGROUND OF THE INVENTION

Among the more effective antibiotics are those that interfere withcommon modes of bacterial gene expression, regulation or activity.Recently, the supercoiling of DNA had been suggested as a possible modeof virulence gene regulation. Local increases or decreases in DNAdensity, due to supercoiling, have been associated with responses tovarious environmental conditions such as, temperature, anaerobiosis, andosmolarity. Appropriate regulation of the accessibility of groups ofgenes to components of the transcriptional apparatus by increasing ordecreasing supercoiling of spacially organized genes may represent aninfecting pathogen's effective response to such environmentalconditions. Enzymes, such as DNA topoisomerases including type 1topoisomerases and DNA gyrases, have been identified which function toeffect the levels of DNA supercoiling. Such enzymes represent usefultargets against which to screen compounds as potential antibiotics.

DNA transformations performed by DNA topoisomerases are accomplished bythe cleavage of either a single strand or both strands. The unit changein the Linking number (Lk) resulting from such transformations is thebest operational distinction between the two classes of topoisomerases(P. O. Brown & N. R. Cozzarelli, Science 206:1081-1083 (1979)). Thelinking number (Lk) is the algebraic number of times one strand crossesthe surface stretched over the other strand. DNA topoisomerases whosereactions proceed via a transient single-stranded break and changing theLk in steps of one are classified as type 1, while enzymes whosereactions proceed via double-stranded breaks and changing the Lk insteps of two are classified as type 2.

Members of type 2 topoisomerase family include DNA gyrase, bacterial DNAtopoisomerase IV, T-even phage DNA topoisomerases, eukaryotic DNAtopoisomerase II, and thermophilic topoisomerase II from Sulfolobusacidocaldarius (see: A. Kikuchi et al., Syst. Appl. Microbiol. 7: 72-78(1986); J. Kato et al., J. Biol. Chem. 267: 25676-25684 (1992); W. M.Huang in DNA Topology and Its Biological Effects (N. R. Cozzarelli andJ. C. Wang, eds., Cold Spring Harbor Laboratory Press, New York, 1990),pp. 265-284; T. -S Hsieh in DNA Topology and Its Biological Effects (N.R. Cozzarelli and J. C. Wang, eds., Cold Spring Harbor Laboratory Press,New York, (1990), pp. 243-263)). The coding sequences of a dozen or sotype 2 enzymes have been determined, and the data suggest that all theseenzymes are evolutionary and structurally related. Topological reactionscatalyzed by type 2 topoisomerases include introduction of negativesupercoils into DNA (DNA gyrase), relaxation of supercoiled DNA,catenation (or decatenation) of duplex circles, knotting and unknottingof DNA.

The family of type 1 topoisomerases comprises bacterial topoisomerase I,E. coli topoisomerase III, S. cerevisiae topoisomerase III (R. A. Kim &J. C. Wang, J. Biol. Chem. 267: 17178-17185 (1992), human topoisomeraseIII (Hanai et al., Proc. Natl. Acad. Sci. 93:3653-3657 (1996)), the type1 topoisomerase from chloroplasts that closely resembles bacterialenzymes (J. Siedlecki et al., Nucleic Acids Res. 11: 1523-1536 (1983),thermophilic reverse gyrases (A. Kikuchi, In DNA: “Topology and ItsBiological Effects” (N. R. Cozzarelli and J. C. Wang, eds., Cold SpringHarbor Laboratory Press, New York, 1990, pp. 285-298); C. Bouthier de laTour et al., J. Bact. 173: 3921-3923 (1991), thermophilic D.amylolyticus topoisomerase III (A. I. Slesarev et al. J. Biol. Chem.266: 12321-12328 (1991), nuclear topoisomerases I and closely relatedenzymes from mitochondria and poxviruses (N. Osheroff, Pharmac. Ther.41: 223-241 (1989)). With respect to the mechanism of catalysis thesetopoisomerases can be divided into two groups. Group A consists ofenzymes that require a divalent cation for activity, and form atransient covalent complex with the 5′-phosphoryl termini (prokaryotictype 1 topoisomerases, S. cerevisiae topoisomerase III, and humantopoisomerase III). Group B includes type 1 topoisomerases that do notrequire a divalent cation for activity, and bind covalently to the3′-phosphoryl termini (nuclear topoisomerases I, enzymes frommitochondria and poxviruses commonly called eukaryotic topoisomerasesI). Type I topoisomerases can carry out the following topologicalreactions: they relax supercoiled DNA (except of reverse gyrases),catenate (or decatenate) single-stranded circular DNAs or duplexesproviding that at least one of the molecules contains a nick or gap, orinteract with single-stranded circles to introduce topological knots(type 1-group A topoisomerases). Reverse gyrase, belonging to type1-group A topoisomerases, is the only topoisomerase shown to be able tointroduce positive supercoils into cDNA.

Research on DNA topoisomerases has progressed from DNA enzymology todevelopmental therapeutics. Bacterial DNA topoisomerase II is animportant therapeutic target of quinolone antibiotics; mammalian DNAtopoisomerase II is the cellular target of many potent antitumor drugs(K. Drlica, Microbiol. Rev. 48: 273-289 (1984) and Biochemistry 27:2253-2259 (1988); B. S. Glisson & W. E. Ross, Pharmacol. Ther. 32:89-106 (1987); A. L. Bodley & L. F. Liu, Biotechnology 6: 1315-1319(1988); L. F. Liu, Annu. Rev. Biochem. 58: 351-375 (1989)). These drugs,referred to as topoisomerase II poisons, interfere with thebreakage-rejoining reaction of type II topoisomerase by trapping a keycovalent reaction intermediate, termed the cleavable complex. Mammaliantopoisomerase I is the cellular target of the antitumor drug topotecan(U.S. Pat. No. 5,004,758), which also traps the covalent reactionintermediate.

As mentioned above, bacterial type I topoisomerases (topoisomerase I &III) are enzymes that alter DNA topology and are involved in a number ofcrucial cellular processes including replication, transcription andrecombination (Luttinger, A., Molecular Microbiol. 15(4): 601-608(1995). These enzymes act by transiently breaking one strand of DNA,passing a single or double strand of DNA through the break and finallyresealing the break. Cleavage of the DNA substrate forms a covalentlinkage between a tyrosine residue of the enzyme and the 5′ end of theDNA chain at the cleavage site (Roca, J. A., TIBS 20:156-160 (1995).

Enzyme inhibition which leads to the stabilization of thecovalent-enzyme-DNA complex (cleavable complex), will invoke chromosomaldamage, and bacterial cell death. Furthermore, this mechanism has thepotential of leading to cell death by virtue of a single inhibitionevent. A small molecular weight inhibitor, which acts by stabilizationof the cleavable complex may act on both topoisomerase I and III becauseof the extensive amino acid sequence similarity between them,particularly in the region of their active sites. The likelihood offuture high level resistance to such agents arising from point mutationmay therefore be low.

Inhibitors of type I topoisomerases, for example, those able tostabilize the protein in a covalent complex with DNA would be lethal orinhibitory to the bacterium and thereby have utility in anti-bacterialtherapy. It is particularly preferred to employ Streptococcal genes andgene products as targets for the development of antibiotics. TheStreptococci make up a medically important genera of microbes. They areknown to produce two types of disease, invasive and toxigenic. Invasiveinfections are characterized generally by abscess formation effectingboth skin surfaces and deep tissues. S. pneumoniae is the second leadingcause of bacteremia in cancer patients. Osteomyelitis, septic arthritis,septic thrombophlebitis and acute bacterial endocarditis are alsorelatively common. There are at least three clinical conditionsresulting from the toxigenic properties of Streptococci. Themanifestation of these diseases result from the actions of exotoxins asopposed to tissue invasion and bacteremia. These conditions include:Streptococcal food poisoning, scalded skin syndrome and toxic shocksyndrome.

SUMMARY OF THE INVENTION

Toward these ends, and others, it is an object of the present inventionto provide polypeptides, inter alia, that have been identified as novelTopoisomerase m by homology between the amino acid sequence set out inFIG. 2 (SEQ ID NO: 2) and known amino acid sequences of other proteinssuch as Bacillus subtilis topoisomerase I which is 25.2% identical and42.2% similar to the sequence in FIG. 2.

It is a further object of the invention, moreover, to providepolynucleotides that encode Topoisomerase III, particularlypolynucleotides that encode the polypeptide herein designated bacterialTopoisomerase III.

In a particularly preferred embodiment of this aspect of the inventionthe polynucleotide comprises the region encoding Topoisomerase imi inthe sequence set out in FIG. 1 (SEQ ID NO: 1).

In another particularly preferred embodiment of the present inventionthere is a novel Topoisomerase III protein from Streptococcus pneumoniaecomprising the amino acid sequence of (SEQ ID NO: 2), or a fragment,analogue or derivative thereof.

In accordance with this aspect of the present invention there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressible by the Streptococcus pneumoniae polynucleotide contained indeposited strain NCIMB deposit number 40794.

In accordance with this aspect of the invention there are providedisolated nucleic acid molecules encoding Topoisomerase III, particularlyStreptococcal Topoisomerase III, including mRNAs, cDNAs, genomic DNAsand, in further embodiments of this aspect of the invention,biologically, diagnostically, clinically or therapeutically usefulvariants, analogs or derivatives thereof, or fragments thereof,including fragments of the variants, analogs and derivatives.

Among the particularly preferred embodiments of this aspect of theinvention are naturally occurring allelic variants of Topoisomerase III.

In accordance with this aspect of the invention there are provided novelpolypeptides of Streptococcal origin referred to herein as TopoisomeraseIII as well as biologically, diagnostically or therapeutically usefulfragments thereof, as well as variants, derivatives and analogs of theforegoing and fragments thereof.

It also is an object of the invention to provide Topoisomerase IIIpolypeptides, particularly bacterial Topoisomerase III polypeptides,that may be employed for therapeutic purposes, for example, to treatdisease, including treatment by conferring host immunity againstbacterial infections, such as Streptococcal infections.

In accordance with yet a further aspect of the present invention, thereis provided the use of a polypeptide of the invention, in particular afragment thereof, for therapeutic or prophylactic purposes, for example,as an antibacterial agent or a vaccine.

In accordance with another aspect of the present invention, there isprovided the use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization.

Among the particularly preferred embodiments of this aspect of theinvention are variants of Topoisomerase III polypeptide encoded bynaturally occurring alleles of the Topoisomerase III gene.

It is another object of the invention to provide a process for producingthe aforementioned polypeptides, polypeptide fragments, variants andderivatives, fragments of the variants and derivatives, and analogs ofthe foregoing.

In a preferred embodiment of this aspect of the invention there areprovided methods for producing the aforementioned Topoisomerase IIIpolypeptides comprising culturing host cells having expressiblyincorporated therein an exogenously-derived Topoisomerase III-encodingpolynucleotide under conditions for expression of Topoisomerase III inthe host and then recovering the expressed polypeptide.

In accordance with another object the invention there are providedproducts, compositions, processes and methods that utilize theaforementioned polypeptides and polynucleotides, inter alia, forresearch, biological, clinical and therapeutic purposes.

In accordance with yet another aspect of the present invention, thereare provided inhibitors of such polypeptides, useful as antibacterialagents. In particular, there are provided antibodies against suchpolypeptides.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are probes that hybridize to bacterialTopoisomerase III sequences useful for detection of bacterial infection.

In certain additional preferred embodiments of this aspect of theinvention there are provided antibodies against Topoisomerase IIIpolypeptides. In certain particularly preferred embodiments in thisregard, the antibodies are selective for Streptococcal TopoisomeraseIII.

In accordance with another aspect of the present invention, there areprovided Topoisomerase III agonists. Among preferred agonists aremolecules that mimic Topoisomerase III, that bind to TopoisomeraseIII-binding molecules, and that elicit or augment TopoisomeraseIII-induced responses. Also among preferred agonists are molecules thatinteract with Topoisomerase III encoding genes or Topoisomerase IIIpolypeptides, or with other modulators of Topoisomerase III activities,and thereby potentiate or augment an effect of Topoisomerase III or morethan one effect of Topoisomerase III and which are also preferablybacteriostatic or bacteriocidal.

In accordance with yet another aspect of the present invention, thereare provided Topoisomerase III antagonists. Among preferred antagonistsare those which bind to Topoisomerase III so as to inhibit the bindingof Topoisomerase III-binding molecules or to stabilize the complexformed between Topoisomerase III and Topoisomerase III binding moleculeto prevent further biological activity arising from the TopoisomeraseIII. Also among preferred antagonists are molecules that bind to orinteract with Topoisomerase III so as to inhibit an effect ofTopoisomerase III or more than one effect of Topoisomerase III or whichprevent expression of Topoisomerase III and which are also preferablybacteriostatic or bacteriocidal.

In a further aspect of the invention there are provided compositionscomprising a Topoisomerase III polynucleotide or a Topoisomerase IIIpolypeptide for administration to cells in vitro, to cells ex vivo andto cells in vivo, or to a multicellular organism. In certain preferredembodiments of this aspect of the invention, the compositions comprise aTopoisomerase III polynucleotide for expression of a Topoisomerase IIIpolypeptide in a host organism to raise an immunological response,preferably to raise immunity in such host against Streptococci orrelated organisms.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill from the following description.It should be understood, however, that the following description and thespecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only. Various changes andmodifications within the spirit and scope of the disclosed inventionwill become readily apparent to those skilled in the art from readingthe following description and from reading the other parts of thepresent disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings depict certain embodiments of the invention. Theyare illustrative only and do not limit the invention otherwise disclosedherein.

FIG. 1 shows the polynucleotide sequence of Streptococcus pneumoniaeTopoisomerase III (SEQ ID NO: 1).

FIG. 2 shows the amino acid sequence of Streptococcus pneumoniaeTopoisomerase III (SEQ ID NO: 2) deduced from the polynucleotidesequence of FIG. 1 (SEQ ID NO: 1).

GLOSSARY

The following illustrative explanations are provided to facilitateunderstanding of certain terms used frequently herein, particularly inthe Examples. The explanations are provided as a convenience and are notlimitative of the invention.

Topoisomerase III-BINDING MOLECULE, as used herein, refers to moleculesor ions which bind or interact specifically with Topoisomerase mpolypeptides or polynucleotides of the present invention, including, forexample enzyme substrates, such as supercoiled DNA, cell membranecomponents and classical receptors. Binding between polypeptides of theinvention and such molecules, including binding or interaction moleculesmay be exclusive to polypeptides of the invention, which is preferred,or it may be highly specific for polypeptides of the invention, which isalso preferred, or it may be highly specific to a group of proteins thatincludes polypeptides of the invention, which is preferred, or it may bespecific to several groups of proteins at least one of which includes apolypeptide of the invention. Binding molecules also include antibodiesand antibody-derived reagents that bind specifically to polypeptides ofthe invention.

GENETIC ELEMENT generally means a polynucleotide comprising a regionthat encodes a polypeptide or a polynucleotide region that regulatesreplication, transcription or translation or other processes importantto expression of the polypeptide in a host cell, or a polynucleotidecomprising both a region that encodes a polypeptide and a regionoperably linked thereto that regulates expression. Genetic elements maybe comprised within a vector that replicates as an episomal element;that is, as a molecule physically independent of the host cell genome.They may be comprised within plasmids. Genetic elements also may becomprised within a host cell genome; not in their natural state but,rather, following manipulation such as isolation, cloning andintroduction into a host cell in the form of purified DNA or in avector, among others.

HOST CELL is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

IDENTITY or SIMILARITY, as known in the art, are relationships betweentwo polypeptide sequences or two polynucleotide sequences, as determinedby comparing the sequences. In the art, identity also means the degreeof sequence relatedness between two polypeptide or two polynucleotidesequences as determined by the match between two strings of suchsequences. Both identity and similarity can be readily calculated(Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).While there exist a number of methods to measure identity and similaritybetween two polynucleotide or two polypeptide sequences, both terms arewell known to skilled artisans (Sequence Analysis in Molecular Biology,von Heinje, G., Academic Press, 1987; Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988).Methods commonly employed to determine identity or similarity betweentwo sequences include, but are not limited to those disclosed inCarillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).Preferred methods to determine identity are designed to give the largestmatch between the two sequences tested. Methods to determine identityand similarity are codified in computer programs. Preferred computerprogram methods to determine identity and similarity between twosequences include, but are not limited to, GCG program package(Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)),BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Mol. Biol. 215: 403(1990)).

ISOLATED means altered “by the hand of man” from its natural state;i.e., that, if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a naturally occurringpolynucleotide or a polypeptide naturally present in a living organismin its natural state is not “isolated,” but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis “isolated”, as the term is employed herein. For example, with respectto polynucleotides, the term isolated means that it is separated fromthe chromosome and cell in which it naturally occurs. As part of orfollowing isolation, such polynucleotides can be joined to otherpolynucleotides, such as DNAs, for mutagenesis, to form fusion proteins,and for propagation or expression in a host, for instance. The isolatedpolynucleotides, alone or joined to other polynucleotides such asvectors, can be introduced into host cells, in culture or in wholeorganisms. Introduced into host cells in culture or in whole organisms,such DNAs still would be isolated, as the term is used herein, becausethey would not be in their naturally occurring form or environment.Similarly, the polynucleotides and polypeptides may occur in acomposition, such as a media formulations, solutions for introduction ofpolynucleotides or polypeptides, for example, into cells, compositionsor solutions for chemical or enzymatic reactions, for instance, whichare not naturally occurring compositions, and, therein remain isolatedpolynucleotides or polypeptides within the meaning of that term as it isemployed herein.

POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. Thus, for instance, polynucleotides as used herein refersto, among others, single- and double-stranded DNA, DNA that is a mixtureof single- and double-stranded regions or single-, double- andtriple-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded, or triple-stranded, or a mixture of single- anddouble-stranded regions. In addition, polynucleotide as used hereinrefers to triple-stranded regions comprising RNA or DNA or both RNA andDNA. The strands in such regions may be from the same molecule or fromdifferent molecules. The regions may include all of one or more of themolecules, but more typically involve only a region of some of themolecules. One of the molecules of a triple-helical region often is anoligonucleotide. As used herein, the term polynucleotide includes DNAsor RNAs as described above that contain one or more modified bases.Thus, DNAs or RNAs with backbones modified for stability or for otherreasons are “polynucleotides” as that term is intended herein. Moreover,DNAs or RNAs comprising unusual bases, such as inosine, or modifiedbases, such as tritylated bases, to name just two examples, arepolynucleotides as the term is used herein. It will be appreciated thata great variety of modifications have been made to DNA and RNA thatserve many useful purposes known to those of skill in the art. The termpolynucleotide as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including inter alia, simple and complex cells. The termpolynucleotide(s) embrace short polynucleotides often referred asoligonucleotides.

POLYPEPTIDES, as used herein, includes all polypeptides as describedbelow. The basic structure of polypeptides is well known and has beendescribed in innumerable textbooks and other publications in the art. Inthis context, the term is used herein to refer to any peptide or proteincomprising two or more amino acids joined to each other in a linearchain by peptide bonds. As used herein, the term refers to both shortchains, which also commonly are referred to in the art as peptides,oligopeptides and oligomers, for example, and to longer chains, whichgenerally are referred to in the art as proteins, of which there aremany types. It will be appreciated that polypeptides often contain aminoacids other than the 20 amino acids commonly referred to as the 20naturally occurring amino acids, and that many amino acids, includingthe terminal amino acids, may be modified in a given polypeptide, eitherby natural processes, such as processing and other post-translationalmodifications, but also by chemical modification techniques which arewell known to the art. Even the common modifications that occurnaturally in polypeptides are too numerous to list exhaustively here,but they are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature, and they arewell known to those of skill in the art. Among the known modificationswhich may be present in polypeptides of the present are, to name anillustrative few, acetylation, acylation, ADP-ribosylation, amidation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination. Such modifications are wellknown to those of skill and have been described in great detail in thescientific literature. Several particularly common modifications,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation, forinstance, are described in most basic texts, such as, for instancePROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton,W. H. Freeman and Company, New York (1993). Many detailed reviews areavailable on this subject, such as, for example, those provided by Wold,F., Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). It will be appreciated, as is well known and as notedabove, that polypeptides are not always entirely linear. For instance,polypeptides may be branched as a result of ubiquitination, and they maybe circular, with or without branching, generally as a result ofposttranslation events, including natural processing event and eventsbrought about by human manipulation which do not occur naturally.Circular, branched and branched circular polypeptides may be synthesizedby non-translation natural process and by entirely synthetic methods, aswell. Modifications can occur anywhere in a polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. In fact, blockage of the amino or carboxyl group in apolypeptide, or both, by a covalent modification, is common in naturallyoccurring and synthetic polypeptides and such modifications may bepresent in polypeptides of the present invention, as well. For instance,the amino terminal residue of polypeptides made in E. coli or othercells, prior to proteolytic processing, almost invariably will beN-formylmethionine. During post-translational modification of thepeptide, a methionine residue at the NH₂-terminus may be deleted.Accordingly, this invention contemplates the use of both themethionine-containing and the methionineless amino terminal variants ofthe protein of the invention. The modifications that occur in apolypeptide often will be a function of how it is made. For polypeptidesmade by expressing a cloned gene in a host, for instance, the nature andextent of the modifications in large part will be determined by the hostcell posttranslational modification capacity and the modificationsignals present in the polypeptide amino acid sequence. For instance, asis well known, glycosylation often does not occur in bacterial hostssuch as, for example, E. coli. Accordingly, when glycosylation isdesired, a polypeptide should be expressed in a glycosylating host,generally a eukaryotic cell. Insect cells often carry out the sameposttranslational glycosylations as do mammalian cells and, for thisreason, insect cell expression systems have been developed to expressefficiently mammalian proteins having native patterns of glycosylation.Similar considerations apply to other modifications. It will beappreciated that the same type of modification may be present in thesame or varying degree at several sites in a given polypeptide. Also, agiven polypeptide may contain many types of modifications. In general,as used herein, the term polypeptide encompasses all such modifications,particularly those that are present in polypeptides synthesized byexpressing a polynucleotide in a host cell.

VARIANT(S) of polynucleotides or polypeptides, as the term is usedherein, are polynucleotides or polypeptides that differ from a referencepolynucleotide or polypeptide, respectively. Variants in this sense aredescribed below and elsewhere in the present disclosure in greaterdetail. With reference to polynucleotides, generally, differences arelimited such that the nucleotide sequences of the reference and thevariant are closely similar overall and, in many regions, identical. Asnoted below, changes in the nucleotide sequence of the variant may besilent. That is, they may not alter the amino acids encoded by thepolynucleotide. Where alterations are limited to silent changes of thistype, a variant will encode a polypeptide with the same amino acidsequence as the reference. Also as noted below, changes in thenucleotide sequence of the variant may alter the amino acid sequence ofa polypeptide encoded by the reference polynucleotide. Such nucleotidechanges may result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptide encoded by the referencesequence, as discussed below. With reference to polypeptides generally,differences are limited so that the sequences of the reference and thevariant are closely similar overall and, in many region, identical. Avariant and reference polypeptide may differ in amino acid sequence byone or more substitutions, additions, deletions, fusions andtruncations, which may be present in any combination.

DESCRIPTION OF THE INVENTION

The present invention relates to novel Topoisomerase III polypeptidesand polynucleotides encoding same, among other things, as described ingreater detail below. In particular, the invention relates topolypeptides and polynucleotides of a novel Topoisomerase III gene ofStreptococcus pneumoniae, which is related by amino acid sequencehomology to Bacillus subtilis topoisomerase I polypeptide. The inventionrelates especially to Streptococcal Topoisomerase III having thenucleotide and amino acid sequences set out in FIG. 1 (SEQ ID NO: 1) andFIG. 2 (SEQ ID NO: 2), and to the Topoisomerase III nucleotide and aminoacid sequences of the DNA isolatable from Deposit No. NC1MB 40794, whichis herein referred to as “the deposited organism” or as the “DNA of thedeposited organism.” It will be appreciated that the nucleotide andamino acid sequences set out in FIG. 1 (SEQ ID NO: 1) and FIG. 2 (SEQ IDNO: 2) were obtained by sequencing the DNA of the deposited organism.Hence, the sequence of the deposited clone is controlling as to anydiscrepancies between it (and the sequence it encodes) and the sequencesof FIG. 1 (SEQ ID NO: 1) and FIG. 2 (SEQ ID NO: 2).

Polynucleotides

In accordance with one aspect of the present invention, there areprovided isolated polynucleotides which encode the StreptococcalTopoisomerase III polypeptide having the deduced amino acid sequence ofFIG. 2 (SEQ ID NO: 2).

Using the information provided herein, such as the polynucleotidesequence set out in FIG. 1 (SEQ ID NO: 1), a polynucleotide of thepresent invention encoding Topoisomerase III polypeptide may be obtainedusing standard cloning and screening procedures. To obtain thepolynucleotide encoding the protein using the DNA sequence given in SEQID NO: 1 typically a library of clones of chromosomal DNA of S.pneumoniae 0100993 in E. coli or some other suitable host is probed witha radiolabelled oligonucleotide, preferably a 17mer or longer, derivedfrom the sequence of FIG. 1. Clones carrying DNA identical to that ofthe probe can then be distinguished using high stringency washes. Bysequencing the individual clones thus identified with sequencing primersdesigned from the original sequence it is then possible to extend thesequence in both directions to determine the full gene sequence.Conveniently such sequencing is performed using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook, J. in MOLECULARCLONING, A Laboratory Manual (2nd edition 1989 Cold Spring HarborLaboratory, see Screening By Hybridization 1.90 and Sequencing DenaturedDouble-Stranded DNA Templates 13.70). Illustrative of the invention, thepolynucleotide set out in FIG. 1 (SEQ ID NO: 1) was discovered in a DNAlibrary derived from Streptococcus pneumoniae 0100993 as described inExample I.

Topoisomerase III of the invention is structurally related to otherproteins of the bacterial Topoisomerase III family, as shown bycomparing the sequence encoding Topoisomerase III from the depositedclone with that of sequence reported in the literature. A preferred DNAsequence is set out in FIG. 1 (SEQ ID NO: 1). It contains an openreading frame encoding a protein of about 147 amino acid residues. Theprotein exhibits greatest homology to Bacillus subtilis topoisomerase Iprotein among known proteins. Topoisomerase III of FIG. 2 (SEQ ID NO: 2)has about 25.2% identity and 42.2% similarity with the amino acidsequence of Bacillus subtilis topoisomerase I.

Polynucleotides of the present invention may be in the form of RNA, suchas mRNA, or in the form of DNA, including, for instance, cDNA andgenomic DNA obtained by cloning or produced by chemical synthetictechniques or by a combination thereof. The DNA may be double-strandedor single-stranded. Single-stranded DNA may be the coding strand, alsoknown as the sense strand, or it may be the non-coding strand, alsoreferred to as the anti-sense strand.

The coding sequence which encodes the polypeptide may be identical tothe coding sequence of the polynucleotide shown in FIG. 1 (SEQ ID NO:1). It also may be a polynucleotide with a different sequence, which, asa result of the redundancy (degeneracy) of the genetic code, encodes thepolypeptide of FIG. 2 (SEQ ID NO: 2).

Polynucleotides of the present invention which encode the polypeptide ofFIG. 2 (SEQ ID NO: 2) may include, but are not limited to the codingsequence for the mature polypeptide, by itself; the coding sequence forthe mature polypeptide and additional coding sequences, such as thoseencoding a leader or secretory sequence, such as a pre-, or pro- orprepro-protein sequence; the coding sequence of the mature polypeptide,with or without the aforementioned additional coding sequences, togetherwith additional, non-coding sequences, including for example, but notlimited to non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription (includingtermination signals, for example), ribosome binding, mRNA stabilityelements, and additional coding sequence which encode additional aminoacids, such as those which provide additional functionalities. The DNAmay also comprise a promoter region which functions to direct thetranscription of the mRNA encoding the Topoisomerase III of thisinvention. Such promoter may be independently useful to direct thetranscription of heterologous gene in recombinant expression system.Furthermore, the polypeptide may be fused to a marker sequence, such asa peptide, which facilitates purification of the fused polypeptide. Incertain embodiments of this aspect of the invention, the marker sequenceis a hexa-histidine peptide, such as the tag provided in the pQE vector(Qiagen, Inc.), among others, many of which are conmercially available.As described in Gentz et al., Proc. Nat'l. Acad. Sci. USA 86: 821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. The HA tag may also be used tocreate fusion proteins and corresponds to an epitope derived ofinfluenza hemagglutinin protein, which has been described by Wilson etal. Cell 37: 767 (1984), for instance.

In accordance with the foregoing, the term “polynucleotide encoding apolypeptide” as used herein encompasses polynucleotides which include asequence encoding a polypeptide of the present invention, particularlybacterial, and more particularly Streptococcus pneumoniaeTopoisomeraseIII having the amino acid sequence set out in FIG. 2 (SEQ ID NO: 2). Theterm encompasses polynucleotides that include a single continuous regionor discontinuous regions encoding the polypeptide (for example,interrupted by integrated phage or insertion sequence or editing)together with additional regions, that also may contain coding and/ornon-coding sequences.

The present invention further relates to variants of the herein abovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 2 (SEQ ID NO: 2). A variant of the polynucleotide may be anaturally occurring variant such as a naturally occurring allelicvariant, or it may be a variant that is not known to occur naturally.Such non-naturally occurring variants of the polynucleotide may be madeby mutagenesis techniques, including those applied to polynucleotides,cells or organisms.

Among variants in this regard are variants that differ from theaforementioned polynucleotides by nucleotide substitutions, deletions oradditions. The substitutions may involve one or more nucleotides. Thevariants may be altered in coding or non-coding regions or both.Alterations in the coding regions may produce conservative ornon-conservative amino acid substitutions, deletions or additions.

Among the particularly preferred embodiments of the invention in thisregard are polynucleotides encoding polypeptides having the amino acidsequence of Streptococcal Topoisomerase III set out in FIG. 2 (SEQ IDNO: 2); variants, analogs, derivatives and fragments thereof.

Further particularly preferred in this regard are polynucleotidesencoding Topoisomerase III variants, analogs, derivatives and fragments,and variants, analogs and derivatives of the fragments, which have theamino acid sequence of Streptococcal Topoisomerase III polypeptide ofFIG. 2 (SEQ ID NO: 2) in which several, a few, 5 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, which do not alter the properties andactivities of Topoisomerase III. Also especially preferred in thisregard are conservative substitutions. Most highly preferred arepolynucleotides encoding polypeptides having the amino acid sequence ofFIG. 2 (SEQ ID NO: 2), without substitutions.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical to a polynucleotide encoding TopoisomeraseIII polypeptide having the amino acid sequence set out in FIG. 2 (SEQ IDNO: 2), and polynucleotides which are complementary to suchpolynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identical toa polynucleotide encoding Topoisomerase III polypeptide of theStreptococcus pneumoniae DNA of the deposited clone and polynucleotidescomplementary thereto. In this regard, polynucleotides at least 90%identical to the same are particularly preferred, and among theseparticularly preferred polynucleotides, those with at least 95% areespecially preferred. Furthermore, those with at least 97% are highlypreferred among those with at least 95%, and among these those with atleast 98% and at least 99% are particularly highly preferred, with atleast 99% being the more preferred.

Particularly preferred embodiments in this respect, moreover, arepolynucleotides which encode polypeptides which retain substantially thesame biological function or activity as the mature polypeptide encodedby the DNA of FIG. 1 (SEQ ID NO: 1).

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the herein above-described polynucleotides. Asherein used, the term “stringent conditions” means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encodingTopoisomerase imi and to isolate cDNA and genomic clones of other genesthat have a high sequence similarity to the Topoisomerase III gene. Suchprobes generally will comprise at least 15 bases. Preferably, suchprobes will have at least 30 bases and may have at least 50 bases.Particularly preferred probes will have at least 30 bases and will have50 bases or less.

For example, the coding region of the Topoisomerase III gene may beisolated by screening using the known DNA sequence to synthesize anoligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the present invention is then used toscreen a library of cDNA, genomic DNA or mRNA to determine which membersof the library to which the probe hybridizes.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsof and diagnostics for disease, particularly human disease, as furtherdiscussed herein relating to polynucleotide assays.

The polynucleotides of the invention that are oligonucleotides, derivedfrom the sequences (SEQ ID NO: 1) may be used as PCR primers in theprocess herein described to determine whether or not the Streptococcuspneumoniae genes identified herein in whole or in part are transcribedin infected tissue. It is recognized that such sequences will also haveutility in diagnosis of the stage of infection and type of infection thepathogen has attained.

The polynucleotides may encode a polypeptide which is the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature polypeptide (when the mature form has more thanone polypeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, may allowprotein transport, may lengthen or shorten protein half-life or mayfacilitate manipulation of a protein for assay or production, amongother things. As generally is the case, in vivo, the additional aminoacids may be processed away from the mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the present invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences which are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Deposited Materials

Streptococcus pneumoliiae 0100993 was deposited at the NationalCollection of Industrial and Marine Bacteria Ltd. (NCIMB), Aberdeen,Scotland under number NCIMB 40794 on Apr. 11, 1996.

The deposit has been made under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Micro-organisms for Purposesof Patent Procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and is not an admission that a deposit is required forenablement, such as that required under 35 U.S.C. §112.

The sequence of the polynucleotides contained in the deposited material,as well as the amino acid sequence of the polypeptide encoded thereby,are controlling in the event of any conflict with any description ofsequences herein.

A license may be required to make, use or sell the deposited materials,and no such license is hereby granted.

Polypeptides

The present invention further relates to a bacterial Topoisomerase IIIpolypeptide that has the deduced amino acid sequence of FIG. 2 (SEQ IDNO: 2).

The invention also relates to fragments, analogs and derivatives ofthese polypeptides. The terms “fragment,” “derivative” and “analog” whenreferring to the polypeptide of FIG. 2 (SEQ ID NO: 2), means apolypeptide which retains essentially the same biological function oractivity as such polypeptide. Fragments derivatives and analogs thatretain at least 90% of the activity of the native Topoisomerase III arepreferred. Fragments derivatives and analogs that retain at least 95% ofthe activity of the native Topoisomerase m are preferred Thus, an analogincludes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide. Incertain preferred embodiments it is a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 2 (SEQ IDNO: 2) may be (i) one in which one or more of the amino acid residuesare substituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be obtained by those ofordinary skill in the art, from the teachings herein.

Among the particularly preferred embodiments of the invention in thisregard are polypeptides having the amino acid sequence of StreptococcalTopoisomerase III set out in FIG. 2 (SEQ ID NO: 2), variants, analogs,derivatives and fragments thereof, and variants, analogs and derivativesof the fragments.

Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids AMa,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gin, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

Further particularly preferred in this regard are variants, analogs,derivatives and fragments, and variants, analogs and derivatives of thefragments, having the amino acid sequence of the Topoisomerase IIIpolypeptide of FIG. 2 (SEQ ID NO: 2), in which several, a few, 5 to 10,1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deletedor added, in any combination. Especially preferred among these aresilent substitutions, additions and deletions, which do not alter theproperties and activities of the Topoisomerase III. Also especiallypreferred in this regard are conservative substitutions. Most highlypreferred are polypeptides having the amino acid sequence of FIG. 2 (SEQID NO: 2) without substitutions.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The polypeptides of the present invention include the polypeptide ofFIG. 2 (SEQ ID NO: 2), in particular the mature polypeptide as well aspolypeptides which have at least 80% identity to the polypeptide of FIG.2 (SEQ ID NO: 2) and preferably at least 90% similarity (more preferablyat least 90% identity) to the polypeptide of FIG. 2 (SEQ ID NO: 2) andmore preferably at least 95% similarity; and still more preferably atleast 95% identity to the polypeptide of FIG. 2 (SEQ ID NO: 2) and alsoinclude portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 contiguous amino acids andmore preferably at least 50 contiguous amino acids.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

Fragments

Also among preferred embodiments of this aspect of the present inventionare polypeptides comprising fragments of Topoisomerase III, mostparticularly fragments of Topoisomerase III having the amino acid setout in FIG. 2 (SEQ ID NO: 2), and fragments of variants and derivativesof the Topoisomerase III of FIG. 2 (SEQ ID NO: 2).

In this regard a fragment is a polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof the aforementioned Topoisomerase III polypeptides and variants orderivatives thereof.

Such fragments may be “free-standing,” i.e., not part of or fused toother amino acids or polypeptides, or they may be comprised within alarger polypeptide of which they form a part or region. When comprisedwithin a larger polypeptide, the presently discussed fragments mostpreferably form a single continuous region. However, several fragmentsmay be comprised within a single larger polypeptide. For instance,certain preferred embodiments relate to a fragment of a TopoisomeraseIII polypeptide of the present comprised within a precursor polypeptidedesigned for expression in a host and having heterologous pre andpro-polypeptide regions fused to the amino terminus of the TopoisomeraseIII fragment and an additional region fused to the carboxyl terminus ofthe fragment. Therefore, fragments in one aspect of the meaning intendedherein, refers to the portion or portions of a fusion polypeptide orfusion protein derived from Topoisomerase III.

Representative examples of polypeptide fragments of the invention,include, for example, may be mentioned those which have from about 5-15,10-20, 15-40, 30-55, 41-75, 41-80, 41-90, 50-100, 75-100, 90-115,100-125, and 110-140 amino acids long.

In this context about includes the particularly recited range and rangeslarger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid ateither extreme or at both extremes.

Among especially preferred fragments of the invention are truncationmutants of Topoisomerase III. Truncation mutants include TopoisomeraseIII polypeptides having the amino acid sequence of FIG. 2 (SEQ ID NO:2), or of variants or derivatives thereof, except for deletion of acontinuous series of residues (that is, a continuous region, part orportion) that includes the amino terminus, or a continuous series ofresidues that includes the carboxyl terminus or, as in double truncationmutants, deletion of two continuous series of residues, one includingthe amino terminus and one including the carboxyl terminus. Fragmentshaving the size ranges set out above also are preferred embodiments oftruncation fragments, which are especially preferred among fragmentsgenerally. Degradation forms of the polypeptides of the invention in ahost cell are also preferred.

Also preferred in this aspect of the invention are fragmentscharacterized by structural or functional attributes of TopoisomeraseIII. Preferred embodiments of the invention in this regard includefragments that comprise alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet-forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions of Topoisomerase III.

Further preferred regions are those that mediate activities ofTopoisomerase III. Most highly preferred in this regard are fragmentsthat have a chemical, biological or other activity of Topoisomerase III,including those with a similar activity or an improved activity, or witha decreased undesirable activity. Routinely one generates the fragmentby well-known methods then compares the activity of the fragment tonature Topoismerase I in a convenient assay such as listed hereinbelow.Highly preferred in this regard are fragments that contain regions thatare homologs in sequence, or in position, or in both sequence and toactive regions of related polypeptides, such as the related polypeptidesset out in FIG. 2 (SEQ ID NO: 2), which include Bacillus subtilistopoisomerase I. Among particularly preferred fragments in these regardsare truncation mutants, as discussed above. Further preferredpolynucleotide fragments are those that are antigenic or immunogenic inan animal, especially in a human.

It will be appreciated that the invention also relates to, among others,polynucleotides encoding the aforementioned fragments, polynucleotidesthat hybridize to polynucleotides encoding the fragments, particularlythose that hybridize under stringent conditions, and polynucleotides,such as PCR primers, for amplifying polynucleotides that encode thefragments. In these regards, preferred polynucleotides are those thatcorrespondent to the preferred fragments, as discussed above.

Vectors, Host Cells, Expression

The present invention also relates to vectors which comprise apolynucleotide or polynucleotides of the present invention, host cellswhich are genetically engineered with vectors of the invention and theproduction of polypeptides of the invention by recombinant techniques.

Host cells can be genetically engineered to incorporate polynucleotidesand express polypeptides of the present invention. Introduction of apolynucleotides into the host cell can be affected by calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction, infection or othermethods. Such methods are described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

Polynucelotide constructs in host cells can be used in a conventionalmanner to produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

In accordance with this aspect of the invention the vector may be, forexample, a plasmid vector, a single or double-stranded phage vector, asingle or double-stranded RNA or DNA viral vector. Plasmids generallyare designated herein by a lower case p preceded and/or followed bycapital letters and/or numbers, in accordance with standard namingconventions that are familiar to those of skill in the art. Startingplasmids disclosed herein are either commercially available, publiclyavailable, or can be constructed from available plasmids by routineapplication of well known, published procedures. Many plasmids and othercloning and expression vectors that can be used in accordance with thepresent invention are well known and readily available to those of skillin the art.

Preferred among vectors, in certain respects, are those for expressionof polynucleotides and polypeptides of the present invention. Generally,such vectors comprise cis-acting control regions effective forexpression in a host operatively linked to the polynucleotide to beexpressed.

Appropriate trans-acting factors either are supplied by the host,supplied by a complementing vector or supplied by the vector itself uponintroduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression. Such specific expression may be inducibleexpression or expression only in certain types of cells or bothinducible and cell-specific. Particularly preferred among induciblevectors are vectors that can be induced for expression by environmentalfactors that are easy to manipulate, such as temperature and nutrientadditives. A variety of vectors suitable to this aspect of theinvention, including constitutive and inducible expression vectors foruse in prokaryotic and eukaryotic hosts, are well known and employedroutinely by those of skill in the art.

A great variety of expression vectors can be used to express apolypeptide of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids, all may be used for expression inaccordance with this aspect of the present invention. Generally, anyvector suitable to maintain, propagate or express polynucleotides toexpress a polypeptide in a host may be used for expression in thisregard.

The appropriate DNA sequence may be inserted into the vector by any of avariety of well-known and routine techniques, such as, for example,those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989).

The DNA sequence in the expression vector is operatively linked toappropriate expression control sequence(s), including, for instance, apromoter to direct mRNA transcription. Representatives of such promotersinclude, but are not limited to, the phage lambda PL promoter, the E.coli lac, trp and tac promoters, the SV40 early and late promoters andpromoters of retroviral LTRs.

In general, expression constructs will contain sites for transcriptioninitiation and termination, and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will include a translationinitiating AUG at the beginning and a termination codon appropriatelypositioned at the end of the polypeptide to be translated.

In addition, the constructs may contain control regions that regulate aswell as engender expression. Generally, in accordance with many commonlypracticed procedures, such regions will operate by controllingtranscription, such as transcription factors, repressor binding sitesand termination, among others.

Vectors for propagation and expression generally will include selectablemarkers and amplification regions, such as, for example, those set forthin Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

Representative examples of appropriate hosts include bacterial cells,such as streptococci, Streptococci, E. coli, streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanomacells; and plant cells.

The following vectors, which are commercially available, are provided byway of example. Among vectors preferred for use in bacteria are pQE70,pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia, and pBR322 (ATCC 37017). Among preferredeukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG availablefrom Stratagene; and pSVK3, pBPV, pMSG and pSVL available fromPharmacia. These vectors are listed solely by way of illustration of themany commercially available and well known vectors that are available tothose of skill in the art for use in accordance with this aspect of thepresent invention. It will be appreciated that any other plasmid orvector suitable for, for example, introduction, maintenance, propagationor expression of a polynucleotide or polypeptide of the invention in ahost may be used in this aspect of the invention.

Promoter regions can be selected from any desired gene using vectorsthat contain a reporter transcription unit lacking a promoter region,such as a chloramphenicol acetyl transferase (“CAT”) transcription unit,downstream of restriction site or sites for introducing a candidatepromoter fragment; i.e., a fragment that may contain a promoter. As iswell known, introduction into the vector of a promoter-containingfragment at the restriction site upstream of the cat gene engendersproduction of CAT activity, which can be detected by standard CATassays. Vectors suitable to this end are well known and readilyavailable, such as pKK232-8 and pCM7. Promoters for expression ofpolynucleotides of the present invention include not only well known andreadily available promoters, but also promoters that readily may beobtained by the foregoing technique, using a reporter gene.

Among known prokaryotic promoters suitable for expression ofpolynucleotides and polypeptides in accordance with the presentinvention are the E. coli laci and lacZ and promoters, the T3 and T7promoters, the gpt promoter, the lambda PR, PL promoters and the trppromoter.

Among known eukaryotic promoters suitable in this regard are the CMVimmediate early promoter, the HSV thymidine kinase promoter, the earlyand late SV40 promoters, the promoters of retroviral LTRs, such as thoseof the Rous sarcoma virus (“RSV”), and metallothionein promoters, suchas the mouse metallothionein-I promoter.

Recombinant expression vectors will include, for example, origins ofreplication, a promoter preferably derived from a highly-expressed geneto direct transcription of a downstream structural sequence, and aselectable marker to permit isolation of vector containing cells afterexposure to the vector.

Polynucleotides of the invention, encoding the heterologous structuralsequence of a polypeptide of the invention generally will be insertedinto the vector using standard techniques so that it is operably linkedto the promoter for expression. The polynucleotide will be positioned sothat the transcription start site is located appropriately 5′ to aribosome binding site. The ribosome binding site will be 5′ to the AUGthat initiates translation of the polypeptide to be expressed.Generally, there will be no other open reading frames that begin with aninitiation codon, usually AUG, and lie between the ribosome binding siteand the initiation codon. Also, generally, there will be a translationstop codon at the end of the polypeptide and there will be apolyadenylation signal in constructs for use in eukaryotic hosts.Transcription termination signal appropriately disposed at the 3′ end ofthe transcribed region may also be included in the polynucleotideconstruct.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous functional regions. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N- or C-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, region also may be added to the polypeptideto facilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stability orto facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilize orpurify polypeptides. For example, EP-A-O 464 533 (Canadian counterpart2045869) discloses fusion proteins comprising various portions ofconstant region of immunoglobulin molecules together with anotherprotein or part thereof. In drug discovery, for example, proteins havebeen fused with antibody Fc portions for the purpose of high-throughputscreening assays to identify antagonists. See, D. Bennett et al.,Journal of Molecular Recognition, 8: 52-58 (1995) and K. Johanson et al.The Journal of Biological Chemistry, 270,(16): 9459-9471 (1995).

Cells typically then are harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Mammalian expression vectors may comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation sites, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking non-transcribedsequences that are necessary for expression. In certain preferredembodiments in this regard DNA sequences derived from the SV40 splicesites, and the SV40 polyadenylation sites are used for requirednon-transcribed genetic elements of these types.

Topoisomerase III polypeptide can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.Well known techniques for refolding protein may be employed toregenerate active conformation when the polypeptide is denatured duringisolation and or purification.

Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

Topoisomerase III polynucleotides and polypeptides may be used inaccordance with the present invention for a variety of applications,particularly those that make use of the chemical and biologicalproperties of Topoisomerase Im. Additional applications relate todiagnosis and to treatment of disorders of cells, tissues and organisms.These aspects of the invention are illustrated further by the followingdiscussion.

Polynucleotide Assays

This invention is also related to the use of the Topoisomerase IIIpolynucleotides to detect complementary polynucleotides such as, forexample, as a diagnostic reagent. Detection of a bacterial TopoisomeraseIII in a eukaryote, particularly a mammal, and especially a human, willprovide a diagnostic method that can add to, define or allow a diagnosisof a disease. Eukaryotes (herein also “individual(s)”), particularlymammals, and especially humans, infected by a Topoisomerase IIIproducing bacterium may be detected at the DNA or RNA level by a varietyof techniques. Nucleic acids for diagnosis may be obtained from anindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Tissue biopsy and autopsy material is also preferred forsamples from an individual to use in a diagnostic assay. The bacterialDNA may be used directly for detection or may be amplified enzymaticallyby using PCR prior to analysis. PCR (Saiki et al., Nature 324: 163-166(1986)). RNA or cDNA may also be used in the same ways. As an example,PCR primers complementary to the nucleic acid encoding Topoisomerase IIIcan be used to identify and analyze Topoisomerase III presence andexpression. Using PCR, characterization of the strain of prokaryotepresent in a eukaryote, particularly a mammal, and especially a human,may be made by an analysis of the genotype of the prokaryote gene. Forexample, deletions and insertions can be detected by a change in size ofthe amplified product in comparison to the genotype of a referencesequence. Point mutations can be identified by hybridizing amplified DNAto radiolabeled Topoisomerase III RNA or alternatively, radiolabeledTopoisomerase III antisense DNA sequences. Perfectly matched sequencescan be distinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Sequence differences between a reference gene and genes having mutationsalso may be revealed by direct DNA sequencing. In addition, cloned DNAsegments may be employed as probes to detect specific DNA segments. Thesensitivity of such methods can be greatly enhanced by appropriate useof PCR or another amplification method. For example, a sequencing primeris used with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic typing of various strains of bacteria based on DNA sequencedifferences may be achieved by detection of alteration inelectrophoretic mobility of DNA fragments in gels, with or withoutdenaturing agents. Small sequence deletions and insertions can bevisualized by high resolution gel electrophoresis. DNA fragments ofdifferent sequences may be distinguished on denaturing formamidegradient gels in which the mobilities of different DNA fragments areretarded in the gel at different positions according to their specificmelting or partial melting temperatures (see, e.g., Myers et al.,Science, 230: 1242 (1985)).

Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase and SI protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Nat'l. Acad. Sci. USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,restriction fragment length polymorphisms (“RFLP”) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations also can be detected by in situ analysis.

Cells carrying mutations or polymorphisms in the gene of the presentinvention may also be detected at the DNA level by a variety oftechniques, to allow for serotyping, for example. Nucleic acids fordiagnosis may be obtained from an infected individual's cells, includingbut not limited to blood, urine, saliva, tissue biopsy and autopsymaterial or from bacteria isolated and cultutered from the abovesources. The bacterial DNA may be used directly for detection or may beamplified enzymatically by using PCR (Saiki et al, Nature, 324:163-166(1986)) prior to analysis. RT-PCR can also be used to detect mutations.It is particularly preferred to used RT-PCR in conjunction withautomated detection systems, such as, for example, GeneScan. RNA or cDNAmay also be used for the same purpose, PCR or RT-PCR. As an example, PCRprimers complementary to the nucleic acid encoding Topoisomerase III canbe made using known methods and used to identify and analyze mutations.For example, deletions and insertions can be detected by a change insize of the amplified product in comparison to the normal genotype.Point mutations can be identified by hybridizing amplified DNA toradiolabeled RNA or alternatively, radiolabeled antisense DNA sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase A digestion or by differences in melting temperatures.These primers may be used for amplifying Topoisomerase III cDNA isolatedfrom a sample derived from an individual. The invention also providessuch primers with 1, 2, 3 or 4 nucleotides removed from the 5′ and/orthe 3′ end. The primers may be used to amplify the gene isolated fromthe individual such that the gene may then be subject to varioustechniques for elucidation of the DNA sequence. In this way, mutationsin the DNA sequence may be detected.

Polypeptide Assays

The present invention also relates to a diagnostic assays such asquantitative and diagnostic assays for detecting levels of TopoisomeraseIII protein in cells and tissues, including determination of normal andabnormal levels. Thus, for instance, a diagnostic assay in accordancewith the invention for detecting expression of Topoisomerase III proteincompared to normal control tissue samples may be used to detect thepresence of an infection. Assay techniques that can be used to determinelevels of a protein, such as an Topoisomerase III protein of the presentinvention, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.Among these ELISAs frequently are preferred. An ELISA assay initiallycomprises preparing an antibody specific to Topoisomerase III,preferably a monoclonal antibody. In addition a reporter antibodygenerally is prepared which binds to the monoclonal antibody. Thereporter antibody is attached a detectable reagent such as radioactive,fluorescent or enzymatic reagent, in this example horseradish peroxidaseenzyme.

Antibodies

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. The present invention includes, for examplesmonoclonal and polyclonal antibodies, chimeric, single chain, andhumanized antibodies, as well as Fab fragments, or the product of an Fabexpression library.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique known in the artwhich provides antibodies produced by continuous cell line cultures canbe used. Examples include various techniques, such as those in Kohler,G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIESAND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice, or other organisms such as other mammals, may be used to expresshumanized antibodies to immunogenic polypeptide products of thisinvention.

Alternatively phage display technology could be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-Fbp or from naive libraries (McCafferty, J.et al., Nature 348, 552-554 (1990); Marks, J. et al., Biotechnology 10:779-783 (1992). The affinity of these antibodies can also be improved bychain shuffling (Clackson, T. et al., Nature 352, 624-628 (1991).

If two antigen binding domains are present each domain may be directedagainst a different epitope—termed ‘bispecific’ antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or purify the polypeptide of thepresent invention by attachment of the antibody to a solid support forisolation and/or purification by affinity chromatography.

Thus among others, antibodies against Topoisomerase III may be employedto inhibit and/or treat infections, particularly bacterial infections,and especially Streptococcal infections as well as to monitor theeffectiveness of antibiotic treatment.

Polypeptide derivatives include antigenically, epitopically orimmunologically equivalent derivatives which form a particular aspect ofthis invention. The term “antigenically equivalent derivative” as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the present invention, interferewith the immediate physical interaction between pathogen and mammalianhost. The term “immunologically equivalent derivative” as used hereinencompasses a peptide or its equivalent which when used in a suitableformulation to raise antibodies in a vertebrate, the antibodies act tointerfere with the immediate physical interaction between pathogen andmammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably the antibody or derivative thereof is modified to make itless immunogenic in the individual. For example, if the individual ishuman the antibody may most preferably be “humanised” ; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody , for example asdescribed in Jones, P. et al. Nature 321: 522-525 (1986) or Tempest etal., Biotechnology 9: 266-273 (1991).

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1:363(1992), Manthorpe et al., Hum. Gene Ther. 1963:4: 419 (1963), deliveryof DNA complexed with specific protein carriers (Wu et al., J. Biol.Chem. 264:16985 (1989), coprecipitation of DNA with calcium phosphate(Benvenisty and Reshef, Proc. Nat'l Acad. Sci. (USA),:83;9551 (1986),encapsulation of DNA in various forms of liposomes (Kaneda et al.,Science 243:375 (1989), particle bombardment (Tang et al, Nature, (1992)356:152, Eisenbraun et al. DNA Cell Biol 12:791(1993) and in vivoinfection using cloned retroviral vectors (Seeger et al., Proc. Nat'lAcad. Sci. (USA) 81:5849 (1984).

Topoisomerase III Binding Molecules and Assays

This invention also provides a method for identification of molecules,such as binding molecules, that bind Topoisomerase III. Genes encodingproteins that bind Topoisomerase III, such as binding proteins, can beidentified by numerous methods known to those of skill in the art, forexample, ligand panning and FACS sorting. Such methods are described inmany laboratory manuals such as, for instance, Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5 (1991).

For instance, expression cloning may be employed for this purpose. Tothis end polyadenylated RNA is prepared from a cell expressingTopoisomerase III, a cDNA library is created from this RNA, the libraryis divided into pools and the pools are transfected individually intocells that are not expressing to Topoisomerase III. The transfectedcells then are exposed to labeled Topoisomerase III. Topoisomerase HIcan be labeled by a variety of well-known techniques including standardmethods of radio-iodination or inclusion of a recognition site for asite-specific protein kinase.) Following exposure, the cells are fixedand binding of Topoisomerase III is determined. These proceduresconveniently are carried out on glass slides.

Pools are identified of cDNA that produced Topoisomerase III-bindingcells. Sub-pools are prepared from these positives, transfected intohost cells and screened as described above. Using an iterativesub-pooling and rescreening process, one or more single clones thatencode the putative binding molecule, such as a binding molecule, can beisolated.

Alternatively a labeled ligand can be photoaffinity linked to a cellextract, such as a membrane or a membrane extract, prepared from cellsthat express a molecule that it binds, such as a binding molecule.Cross-linked material is resolved by polyacrylamide gel electrophoresis(“PAGE”) and exposed to X-ray film. The labeled complex containing theligand-binding can be excised, resolved into peptide fragments, andsubjected to protein microsequencing. The amino acid sequence obtainedfrom microsequencing can be used to design unique or degenerateoligonucleotide probes to screen cDNA libraries to identify genesencoding the putative binding molecule.

Polypeptides of the invention also can be used to assess TopoisomeraseIII binding capacity of Topoisomerase III binding molecules, such asbinding molecules, in cells or in cell-free preparations.

Polypeptides of the invention may also be used to assess the binding orsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics.

Antagonists and Agonists—Assays and Molecules

As mentioned above, both increases and decreases in DNA density havebeen associated with bacterial responses to environmental challenges.Accordingly, modulating, i.e., agonizing or antagonizing, theappropriate response could result in a potential antibiotic effect.

The invention also provides a method of screening compounds to identifythose which enhance or block the action of Topoisomerase III on cells,such as its interaction with substrate molecules, such as supercoiledDNA. Compounds which block the action of Topoisomerase III on cellsinclude those which act as poisons and stabilize Topoisomerase III in acovalent complex with DNA, resulting in an inhibitory effect on cellgrowth. An antagonist is a compound which decreases the naturalbiological functions of Topoisomerase III. An agonist is a compoundwhich increases the natural biological functions of Topoisomerase III.

Barrett et al., Antimicrob. Agents Chemother. 34:1 (1990) reviewin-vitro assays which can be used to measure inhibition oftopoisomerases. These assays can be categorized as catalytic assays andnoncatalytic assays. Catalytic assays for bacterial topoisomerase IIIinclude, for example, assays to measure the relaxation of supercoiledDNA. Noncatalytic assays, also known as ‘cleavable complex’ assays,measure the formation of a key covalent reaction intermediate.Froelich-Ammon and Osheroff J. Biol. Chem. 270:21429 (1995) review themechanistic basis of noncatalytic assays of topoisomerase poisons.

Supercoiled DNA Relaxation Assay

To screen for inhibitors of the relaxation reaction, a candidateinhibitor and a preparation of Topoisomerase III are incubated with asupercoiled DNA substrate, for example plasmid or phage DNA, in anappropriate buffer containing Mg²⁺, or an alternative divalent metalion. Reaction products are separated by agarose gel electrophoresis,visualized by ethidium bromide staining, and quantified by densitometry.

DNA Oligomer Cleavage Assay

A single stranded DNA oligomer containing appropriate cleavage sites,for example the 22mer GAATGAGCCGCAACTTCGGGAT (SEQ ID NO: 3), or anappropriately labelled derivative, may be used as substrate. Anappropriate label may be a radiolabel or a fluorescent chromophoreattached at the 5′ or 3′ end of the oligo, according to the specificassay used. The substrate is incubated with a candidate inhibitor and apreparation of Topoisomerase III, in an appropriate buffer. The buffermay contain Mg²⁺ or an alternative divalent metal ion. Mg²⁺ is notessential for the cleavage reaction, although its inclusion may bedesirable to facilitate the interaction of certain classes ofinhibitors. The reaction is stopped by the addition of an appropriatedenaturant, for example 1% SDS or 100 mM NaOH. Generation of thecleavable complex (stabilization of the key covalent reactionintermediate) may be measured by a number of methods. For example,electrophoresis using a denaturing polyacrylamide gel can be used toseparate the 5′ labelled cleaved DNA product which may then bequantified by densitometry. Alternatively, the 3′ labelled DNA productmay be assayed by virtue of its covalent association with TopoisomeraseIII. This may be performed by the SDS/K precipitation assay, in whichradiolabelled DNA associated with precipitated protein is measured, orby a capture assay format in which Topoisomerase III is immobilizedusing an antibody and the amount of associated labelled DNA is measured.

Whole Cell Assays

Topoisomerase III-like effects of potential agonists and antagonists andpoisons, may by measured, for instance, by determining activity of areporter system that is sensitive to alterations in gene expressionfollowing interaction of the candidate molecule with a cell orappropriate cell preparation. Reporter systems that may be useful inthis regard include but are not limited to colorimetric labeledsubstrate converted into product, a reporter gene that is responsive tochanges in Topoisomerase III activity, and binding assays known in theart.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity, or stabilize the keycovalent reaction intermediate with DNA. Potential antagonists also maybe small organic molecules, a peptide, a polypeptide such as a closelyrelated protein or antibody that binds the same sites on a bindingmolecule, such as a binding molecule, without inducing TopoisomeraseIII-induced activities, thereby preventing the action of TopoisomeraseIII by excluding Topoisomerase III from binding.

Potential antagonists include a small molecule which binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such as binding molecules, such thatnormal biological activity is prevented. Examples of small moleculesinclude but are not limited to small organic molecules, peptides orpeptide-like molecules.

Other potential antagonists include antisense molecules. Antisensetechnology can be used to control gene expression through antisense DNAor RNA or through double- or triple-helix formation. Antisensetechniques are discussed, for example, in—Okano, J. Neurochem. 56: 560(1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENEEXPRESSION, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance Lee et al. Nucleic Acids Research 6: 3073(1979); Cooney et al., Science 241: 456 (1988); and Dervan et al.,Science 251: 1360 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA. For example, the 5′ codingportion of a polynucleotide that encodes the mature polypeptide of thepresent invention may be used to design an antisense RNA oligonucleotideof from about 10 to 40 base pairs in length. A DNA oligonucleotide isdesigned to be complementary to a region of the gene involved intranscription thereby preventing transcription and the production ofTopoisomerase III. The antisense RNA oligonucleotide hybridizes to themRNA in vivo and blocks translation of the mRNA molecule intoTopoisomerase III polypeptide. The oligonucleotides described above canalso be delivered to cells such that the antisense RNA or DNA may beexpressed in vivo to inhibit production of Topoisomerase III.

Preferred potential antagonists include compounds related to andderivatives of each of the DNA sequences provided herein may be used inthe discovery and development of antibacterial compounds. The encodedprotein upon expression can be used as a target for the screening ofantibacterial drugs. Additionally, the DNA sequences encoding the aminoterminal regions of the encoded protein or Shine-Delgarno or othertranslation facilitating sequences of the respective mRNA can be used toconstruct antisense sequences to control the expression of the codingsequence of interest.

The antagonists and agonists of the invention may be employed in acomposition with a pharmaceutically acceptable carrier, e.g., ashereinafter described.

The antagonists and agonists may be employed for instance to inhibitStreptococcal infections.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with Topoisomerase III, or aantigenic fragment or variant thereof, adequate to produce antibody toprotect said individual from infection, particularly bacterial infectionand most particularly Streptococcal infection. Yet another aspect of theinvention relates to a method of inducing immunological response in anindividual which comprises, through gene therapy, delivering geneencoding Topoisomerase III, or a antigenic fragment or a variantthereof, for expressing Topoisomerase III, or a fragment or a variantthereof in vivo in order to induce an immunological response to produceantibody to protect said individual from disease.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into a host capable or having inducedwithin it an immunological response, induces an immunological responsein such host to a Topoisomerase III or protein coded therefrom, whereinthe composition comprises a recombinant Topoisomerase III or proteincoded therefrom comprising DNA which codes for and expresses an antigenof said Topoisomerase III or protein coded therefrom.

The Topoisomerase III or a fragment thereof may be fused with co-proteinwhich may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such asGlutathione-S-transferase (GST) or beta-galactosidase, relatively largeco-proteins which solubilise the protein and facilitate production andpurification thereof. Moreover, the co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system.The co-protein may be attached to either the amino or carboxy terminusof the first protein.

The present invention also includes a vaccine formulation whichcomprises the immunogenic recombinant protein together with a suitablecarrier. Since the protein may be broken down in the stomach, it ispreferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation instonic with the bodily fluid, preferably the blood, of theindividual; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

Whilst the invention has been described with reference to certainTopoisomerase III, it is to be understood that this covers fragments ofthe naturally occurring protein and similar proteins (for example,having sequence homologies of 50% or greater) with additions, deletionsor substitutions which do not substantially affect the immunogenicproperties of the recombinant protein.

Compositions

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or the agonists or antagonists.Thus, the polypeptides of the present invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to a subject. Such compositions comprise,for instance, a media additive or a therapeutically effective amount ofa polypeptide of the invention and a pharmaceutically acceptable carrieror excipient. Such carriers may include, but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol and combinationsthereof. The formulation should suit the mode of administration.

Kits

The invention further relates to diagnostic and pharmaceutical packs andkits comprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for humanadministration.

Administration

Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

The pharmaceutical compositions generally are administered in an amounteffective for treatment or prophylaxis of a specific indication orindications. In general, the compositions are administered in an amountof at least about 10 μg/kg body weight. In most cases they will beadministered in an amount not in excess of about 8 mg/kg body weight perday. Preferably, in most cases, dose is from about 10 μg/kg to about 1mg/kg body weight, daily. It will be appreciated that optimum dosagewill be determined by standard methods for each treatment modality andindication, taking into account the indication, its severity, route ofadministration, complicating conditions and the like.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters,etc.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent Streptococcal wound infections.

Many orthopedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteraemia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response.

A suitable unit dose for vaccination is 0.5-5 microgran/kg of antigen,and such dose is preferably administered 1-3 times and with an intervalof 1-3 weeks.

With the indicated dose range, no adverse toxicological effects will beobserved with the compounds of the invention which would preclude theiradministration to suitable individuals.

The antibodies described above may also be used as diagnostic reagentsto detect the presence of bacteria containing Topoisomerase.

Each reference disclosed herein is incorporated by reference herein inits entirety. Any patent application to which this application claimspriority is also incorporated by reference herein in its entirety.

In order to facilitate understanding of the following example certainfrequently occurring methods and/or terms will be described.

EXAMPLES

The present invention is further described by the following examples.The examples are provided solely to illustrate the invention byreference to specific embodiments. These exemplification's, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

Certain terms used herein are explained in the foregoing glossary.

All examples were carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

All parts or amounts set out in the following examples are by weight,unless otherwise specified.

Unless otherwise stated size separation of fragments in the examplesbelow was carried out using standard techniques of agarose andpolyacrylamide gel electrophoresis (“PAGE”) in Sambrook et al.,MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989) and numerous otherreferences such as, for instance, by Goeddel et al., Nucleic Acids Res.8: 4057 (1980).

Unless described otherwise, ligations were accomplished using standardbuffers, incubation temperatures and times, approximately equimolaramounts of the DNA fragments to be ligated and approximately 10 units ofT4 DNA ligase (“ligase”) per 0.5 microgram of DNA.

The polynucleotide having the DNA sequence given in (SEQ ID NO: 1) wasobtained from the sequencing of a library of clones of chromosomal DNAof Streptococcus pneumoniae 0100993 in E. coli.

To obtain the polynucleotide encoding the Topoisomerase III proteinusing the DNA sequence given in (SEQ ID NO: 1) typically a library ofclones of chromosomal DNA of Streptococcus pneumoniae 0100993 in E. colior some other suitable host is probed with a radiolabelledoligonucleotide, preferably a 17mer or longer, derived from the partialsequence. Clones carrying DNA identical to that of the probe can then bedistinguished using high stringency washes. By sequencing the individualclones thus identified with sequencing primers designed from theoriginal sequence it is then possible to extend the sequence in bothdirections to determine the full gene sequence. Conveniently suchsequencing is performed using denatured double stranded DNA preparedfrom a plasmid clone. Suitable techniques are described by Maniatis, T.,Fritsch, E. F. and Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, New York (1989). (see Screening By Hybridization 1.90 andSequencing Denatured Double-Stranded DNA Templates 13.70).

Example 1

Isolation of DNA coding for Novel Topoisomerase III Protein fromStreptococcus pneumoniae

The polynucleotide having the DNA sequence given in (SEQ ID NO: 1) wasobtained from a library of clones of chromosomal DNA of Streptococcuspneumoniae in E.coli. In some cases the sequencing data from two or moreclones containing overlapping Streptococcus pneumoniae DNA was used toconstruct the contiguous DNA sequence in (SEQ ID NO: 1). Libraries maybe prepared Libraries may be prepared by routine methods, for example,Methods 1 and 2 below.

Total cellular DNA is isolated from Streptococcus pneumoniae strain0100993 according to standard procedures and size-fractionated by eitherof two methods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E.coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolysed with a combination of fourrestriction enzymes (RsaI, PalI, AluI and Bsh12351) andsize-fractionated according to standard procedures. EcoRI linkers areligated to the DNA and the fragments then ligated into the vector LambdaZapII that have been cut with EcoRI, the library packaged by standardprocedures, and E.coli infected with the packaged library. The libraryis amplified by standard procedures.

3 442 base pairs nucleic acid single linear Genomic DNA not provided 1GATGTTCGTT GAAGCCGAAC TGAAGCCGAC CGGTCTGGAG CGAGCCGAAG CCCAGGCCAT 60CGCCGACGCG GTGCGCAACC AGACCGGCAC CGTCACCGAA GAAGCCAAGC CCAGCACGCA 120GGCCAGCCCG TTGCTGTACG ACCTGACCAC GCTGCAACGC GAAGCCAACT CGCGCTTCGG 180CTTCAGCGCC AAGACCACGC TGTCGCTGGC ACAGGCGCTA TACGAAAAAC ACAAGGCGCT 240GACCTACCCG CGGACCGATT CACGCGCCCT GCCTGAAGAC TACCTGGGCG TGGTCAAGCA 300GACCGTCGGC GTGCTGGCCG AGGGCGACCT GCCCGGCCCG CTGAAGGCTT TGTCGGCTCA 360CGCCGAAAAG GCGCTGAAAG AGGGTTACAT CAAACCCAAC AAGCGCGTGT TCGACAACGC 420CAAGGTGTCG GACCACTTCG CC 442 147 amino acids amino acid single linearprotein not provided 2 Met Phe Val Glu Ala Glu Leu Lys Pro Thr Gly LeuGlu Arg Ala Glu 1 5 10 15 Ala Gln Ala Ile Ala Asp Ala Val Arg Asn GlnThr Gly Thr Val Thr 20 25 30 Glu Glu Ala Lys Pro Ser Thr Gln Ala Ser ProLeu Leu Tyr Asp Leu 35 40 45 Thr Thr Leu Gln Arg Glu Ala Asn Ser Arg PheGly Phe Ser Ala Lys 50 55 60 Thr Thr Leu Ser Leu Ala Gln Ala Leu Tyr GluLys His Lys Ala Leu 65 70 75 80 Thr Tyr Pro Arg Thr Asp Ser Arg Ala LeuPro Glu Asp Tyr Leu Gly 85 90 95 Val Val Lys Gln Thr Val Gly Val Leu AlaGlu Gly Asp Leu Pro Gly 100 105 110 Pro Leu Lys Ala Leu Ser Ala His AlaGlu Lys Ala Leu Lys Glu Gly 115 120 125 Tyr Ile Lys Pro Asn Lys Arg ValPhe Asp Asn Ala Lys Val Ser Asp 130 135 140 His Phe Ala 145 22 basepairs nucleic acid single linear Genomic DNA not provided 3 GAATGAGCCGCAACTTCGGG AT 22

What is claimed is:
 1. An isolated polypeptide comprising SEQ ID NO:2,wherein the isolated polypeptide exhibits topoisomerase activity.
 2. Acomposition comprising the isolated polypeptide of claim 1 and anacceptable carrier.
 3. The isolated polypeptide of claim 1, wherein theisolated polypeptide further comprises a heterologous amino acidsequence.
 4. A composition comprising the isolated polypeptide of claim3 and an acceptable carrier.
 5. The isolated polypeptide of claim 1,wherein the isolated polypeptide consists of SEQ ID NO:2, wherein theisolated polypeptide exhibits topoisomerase activity.
 6. A compositioncomprising the isolated polypeptide of claim 5 and an acceptablecarrier.
 7. An isolated polypeptide comprising at least 50 consecutiveamino acids of SEQ ID NO:2, wherein the isolated polypeptide exhibitstopoisomerase activity.
 8. A composition comprising the isolatedpolypeptide of claim 7 and an acceptable carrier.
 9. The isolatedpolypeptide of claim 7, wherein the isolated polypeptide furthercomprises a heterologous amino acid sequence.
 10. A compositioncomprising the isolated polypeptide of claim 9 and an acceptablecarrier.
 11. An isolated polypeptide comprising at least 30 consecutiveamino acids of SEQ ID NO:2, wherein the isolated polypeptide exhibitstopoisomerase activity.
 12. A composition comprising the isolatedpolypeptide of claim 11 and an acceptable carrier.
 13. The isolatedpolypeptide of claim 11, wherein the isolated polypeptide furthercomprises a heterologous amino acid sequence.
 14. A compositioncomprising the isolated polypeptide of claim 13 and an acceptablecarrier.